The present invention relates to a method for reducing the serum separation/syneresis in a pectin containing aqueous mass by the use of certain enzymes, and enzyme compositions therefore.
Dicotyledonous plants comprise some of the major crops cultured by man, such as beans, peas, beets as well as most other fruits and vegetables.
The primary cell wall and middle lamella of these plants usually have a high content of pectic substances. The pectic matrix consists of smooth regions of polygalacturonic acids and of rhamnogalacturonan.
The polygalacturonic acid areas are usually highly esterified by methoxyl groups. Side groups consisting of araban, galactan and arabinogalactan are attached to the rhamnogalacturonan residues. The presence and distribution of the carboxy-methyl groups in pectin significantly alters its solubility and physico-chemical and gel forming properties.
This is of major importance both in the plant cell wall, where the pectic matrix is a key regulator of porosity and passive diffusion of macromolecules but also in industrial processing of pectin and pectin containing plant material.
Pectinesterase (PE), EC 3.1.1.11 (Enzyme Nomenclature 1992, Academic Press, Inc., 1992), hydrolyses the ester linkage between methanol and galacturonic acid in esterified pectin. PE is found both in plants and in microorganisms. That is, PE offers a possibility of completely controlling the properties of pectin by altering the degree of esterification (DE).
Thus, an endogenous highly methoxylated content of pectin (HM pectin) in various fruits and vegetables can enzymatically be modified to a low methoxylated pectin by pectinesterase. In combination with the natural content of calcium ions this is sufficient for an in situ gelation or an in situ thickening to take place if the PE is substantially free from depolymerizing enzyme activities (Calesnik, E. J. et al 1950, Arch. of Biochem., 29, 432-440. Meurens, M., 1978, Rev. Ferment. Ind. Aliment., 33, 95-104), since the presence of such activities will substantially break down the pectin.
It is a well known problem that several products that are fully or partially vegetable or fruit based, such as ketchup, mustard and jam, xe2x80x9cweepxe2x80x9d or separate on standing. The processes used result in an indelicate appearance of the product. Furthermore use of the product on e.g. bread or rolls results in the bread or roll getting soaked and disintegrate. Similarly other products such as fruit soups and yoghurts separate and develop an unattractive appearance.
It is an object of this invention to provide a method of causing a decrease of the serum separation in an aqueous mass containing methoxylated pectin without the need for addition of stabilising agents, such as externally added hydrocolloids, modified starch etc.
The present invention relates to a method for reducing the serum separation of an aqueous mass containing pectin, comprising the steps of
(a) providing an aqueous mass substantially free from pectin depolymerizing enzymes.
(b) adding an effective amount of pectinesterase, and
(c) incubating said mass in the presence of divalent cations.
Surprisingly it has been found that no other stabilising agent is needed in relation to the method according to the invention.
Hereby the method according to the invention offers the unique feature that the serum separation can be reduced or even avoided without the addition of any further external agents, whereby the only external agent being added is an effective amount of pectinesterase, and optionally divalent ions, such as alkaline earth ions, especially calcium ions.
Thus, for the first time it has been proven possible to utilise deesterified endogenous pectin in itself for a direct reduction of serum separation.
As indicated the present invention relates to a method for reducing the serum separation of an aqueous mass containing pectin, comprising the steps of:
(a) providing an aqueous mass substantially free from pectin depolymerizing enzymes.
(b) adding an effective amount of pectinesterase, and
(c) incubating said mass in the presence of divalent cations.
According to one embodiment of the invention the pectin may be endogenous HM.
In an embodiment of the method according to the invention the pectinesterase used is derived from a fungus of the genus Aspergillus, preferably A. japonicus, (S. Ishii et al., 1979, Journal of Food Science 44, p 611-614), A. aculeatus , A. niger (EP 0 388 593 A1), A. awamori (EP 0 388 593 A1), or the genera Fusarium, Sclerotonia, or Penicillium, (Kikkoman: DE 2843351; U.S. Pat. No. 4,200,694). These pectinesterases exhibit a relatively low pH optimum, corresponding to the relatively low pH optimum of many fruits.
The pectinesterase to be used in the invention should preferably be substantially free from pectin depolymerizing enzymes, such as pectinlyases or polygalacturonases. In case these activities are present in substantial amounts the pectin will be degraded.
Such enzymes are obtainable by using a host system for the expression of the enzyme which does not produce any pectin depolymerizing enzymes (WO 94/25575).
The activity of the pectinesterase is indicated in Pectin Esterase Units (PEU) defined as the amount of enzyme which under standardised conditions hydrolyses 1 mmol carboxyl groups per minute. A folder describing the Novo Nordisk assay ABT-SM-0005.02.1 is available upon request.
According to the invention the pectin should be present in the aqueous mass in an amount from 0.1% w/w to 10% w/w preferably from 0.2% w/w to 1.0% w/w.
In further embodiments of the invention it may be advantageous to add sugar, acetic acid and/or salt in amounts from 0-60% w/w, 0-10% w/w, and 0-10% w/w, respectively.
The method of the invention will normally be carried out at a pH value from 2.5 to 7.5, preferably from pH 3 to pH 5, and for a time period for the incubation step from 5 to 60 minutes, preferably from 10 seconds to 30 minutes.
The incubation step will normally be performed at a temperature from 10 to 60xc2x0 C., preferably from 15 to 50xc2x0 C.
According to the invention the PE is added in an amount of from 4 to 400 PEU, preferably from 9 to 135 PEU, better from 25 to 110 PEU, still better from 35 to 90 PEU, and more preferably from 45 to 70 PEU/kg VSS (Vegetable Soluble Solids, VSS is measured refractometrically as % Brix).
According to the invention the method is designed for use with a pertinacious mass, especially food, and especially products such as tomato juice, tomato slurry, tomato paste or salsa or ketchup.
The method of the invention has been found to be advantageous with products wherein the pectin containing aqueous mass is derived from broccoli, pepper, mustard, apples, tomatoes oranges, lemons, grapes, lime, pears, carrots, peas, cauliflower, and berries, such as blackcurrant, blue-berries, strawberries, and raspberries.
Products obtained by the process of the invention are such as jam, marmalade, jelly, juice, paste, soup, dressing, sauce, condiment, ketchup, salsa, chutney, pudding, mousse, or other deserts.
Materials:
Hot break tomato paste
Pectinesterase prepared according to WO 94/25575
Brookfield Viscometer, DVII
DUR Refractometer, Schmidt and Haensch, Germany
Blotter test paper (Bridge and Company)
Methods:
Viscosity Measurement:
The viscosity was measured by Spindel C, specification no. 93. Measurements at shear rate 2.5 rpm and 20 rpm were carried out.
Measurement of Vegetable Soluble Solids (VSS):
A drop of the test solution is applied to the sample well in the DUR refractometer.
The instrument indicates directly the solids content in % Brix.
Blotter Test:
To quantify the serum separation from the solid content the serum separation evaluation procedure of using a blotter test paper was used (Gould W.A., et al 1992, Tomato Production Processing and Technology 3rd edition CTI Publications).
5 7.0 g of ketchup was weighted out in a metal cylinder (dia 38 mm) placed in the centre of the blotter paper. After 7 min the serum separation was measured at the four rules. The measurement is given as an average of the four figures.
A low value indicates a low degree of syneresis/serum separation.